Enzyme-passage free culture of mouse embryonic stem cells on thermo-responsive polymer surfaces

Abstract

Cell based therapies offer potentially revolutionary treatments for a number of diseases, but are dependent on the culture and supply of defined cell types in appropriate numbers. In turn, this supply requires changes to current cell culture passaging methods, which commonly use enzymatic digestion.Here we demonstrate, for the first time, the use of thermoresponsive poly (methoxyethoxyethylmethacrylate-co-oligoethyleneglycolmethacrylate) (poly(MEO 2 MA-co-OEGMA)) as a responsive surface on which to culture embryonic stem cells (mouse; used as a model embryonic stem cell type). Thermo-responsive copolymer brushes composed of MEO 2 MA and OEGMA were grafted from hydroxyl plasma-polymer functionalised glass slides using atom transfer radical polymerisation (ATRP) and their thermo-responsive behaviour characterised. Feeder-free mouse embryonic stem cells (mESCs) were seeded on these surfaces following adsorption of fibronectin (to encourage cell attachment) and cultured for three days. Subsequent passaging experiments were performed over 10 passage cycles and the cells analysed for population doubling times and retention of the stem cell geno/phenotype. These thermoresponsive polymer/fibronectin surfaces were found to be suitable for mESC culture without promoting differentiation during the early stages of culture. This indicated that these materials have promise as a new generation of culture materials for enzyme free cell culture and passage suitable for generating cell populations for clinical applications.

Upper critical solution temperature thermo-responsive polymer brushes and a mechanism for controlled cell attachment

“…[12][13][14] Thermoresponsive co-polymers from poly(ethyleneglycol)methacrylate monomers have also been attached to, or grown from, surfaces in the form of polymer brushes, allowing control of cell attachment and detachment at temperature close to 37 C. [15][16][17] Similar approaches have been adopted to control cell attachment with polypeptide brushes based on L-alanine derivatives with LCST-type thermoresponsive behaviour, for which capture and release of MCF-7 cells over the temperature range from 37 C to 20 C was demonstrated. 18 However, there has been relatively little work focused on polymer brushes with Upper Critical Solution Temperature (UCST)-type phase transition behaviour which might allow controlled cell attachment at temperatures below 37 ⁰C but cell release above this temperature.…”

Thermoresponsive magnetic colloidal gels via surface-initiated polymerisation from functional microparticles

“…On reduction of the suspension temperature to below the expected lower critical solution temperature of PDEGMA (<25°C), the polymer chains became hydrated and chain-extended, resulting in cell detachment from the microparticle surfaces. This phenomenon of thermoreversible bioadhesion hasbeen observed previously with grafted PEGMA polymers on various substrates, [41][42][43][44][45][46] Presto Blue viability assays with 3T3 fibroblasts cultured within the prepared colloidal gel showed that the cells proliferated and remained viable with significant increases in cell number over time from Figure S8 ESI) that the proliferation capacity on the 2D surface was lower than in the 3D gel. The total number of cells from the 3D matrices was higher compared to that for cells grown on the 2D plastic surfaces.…”

Polymer Brushes with Precise Architectures for Molecular Biorecognition

“…The behavior was controlled by temperature cycles around the LCST of the macromolecular platform. In a similar fashion, Dey et al described the use of these thermorresponsive copolymer brushes as responsive platforms for culturing mouse embryonic stem cells (mESCs) and the control of their attachment/detachment to the substrate [22]. Wei et al demonstrated the ability of these surfaces to confer an osteogenetic capacity to the modified substrates [23].…”